ABSTRACT Millions of people suffer from xerostomia, or ?drymouth? resulting from lack of saliva, producing a decreased quality of life due to increased dental caries, oropharyngeal infections, difficulties with swallowing (dysphagia) and digestion (mucositis), loss of taste, and pain. Regenerative medicine can offer innovative strategies capable of restoring gland function in patients that have few alternatives. However, there is a current lack of basic scientific knowledge regarding the mechanisms of gland regeneration and of the ability of scaffolds to promote this process, which remains a substantial limitation in development of therapeutics. In prior work, we developed nanofiber scaffolds that support the attachment, survival, and apicobasal polarization of salivary epithelial cells in vitro, which is a requirement for secretory function. Additionally, micropatterning of the scaffold with hemispherical wells promoted epithelial cell structure and function. Since the secretory acinar cell phenotype is lost when primary mouse submandibular salivary gland epithelial cells are grown in culture either in the presence or absence of nanofiber scaffolds, we investigated the requirement for mesenchymal cells in maintaining their phenotype. Primary salivary gland mesenchyme cells, but not an embryonic mesenchyme cell line, maintained acinar differentiation in co-cultures. Mesenchymal factors were able to substitute for the mesenchyme to maintain acinar differentiation of primary epithelial cells. These mesenchymal factors, when incorporated into a scaffold, may support acinar differentiation. This application proposes an innovative, multidisciplinary strategy to engineer nanofiber scaffolds that are integrated with a porous polymeric ?sponge?- like underlayer that will recruit vasculature and facilitate delivery, survival and differentiation of transplanted cells in vivo. We hypothesize that a nanofiber scaffold functionalized with mesenchymal factors and integrated with a sponge underlayer will enable transplantation of progenitor/proacinar cells while facilitating integration with the host mesenchyme and vasculature to restore salivary function in vivo. The functionalized nanofiber surface will deliver the epithelial progenitor cells and support retention of proacinar differentiation. Functionalization of the sponge with angiogenic factors will recruit and facilitate assembly of vascular networks to promote integration with the host and effective regeneration of functional tissue in vivo. The scaffolds will be tested in a preclinical mouse salivary gland resection model to examine efficacy in supporting tissue regeneration in vivo. Animals will be assessed for salivary flow and saliva quality, tissue regrowth, differentiation state of cells within the new growth, and integration of the regenerated tissue with the host vascular system. The studies proposed here using a small animal preclinical model will inform future testing of an optimized scaffold in a large animal model, leading to clinical application. Abbreviations: Aqp5 (Aquaporin 5), DA (diacrylate) DAPI (4',6-diamidino-2-phenylindole), EC (endothelial cell), E-Cad (E-cadherin), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), EMT (epithelial- mesenchymal transition), epidermal growth factor (EGF), FACS (fluorescent activated cell sorting), FFPE (formalin-fixed, paraffin-embedded), FGF (fibroblast growth factor), FTIR (Fourier transform infrared spectroscopy), H&E (hematoxylin and eosin), ICC (immunocytochemistry), IHC (immunohistochemistry), MA (methacrylate), MACS (magnetic bead activated cell sorting), Mx-ICC (multiplexed immunocytochemistry), OCT (Optimal Cutting Temperature Compound), N-hydroxysuccinimide (NHS), PEG (Poly ethylene glycol), PGS (poly(glycerol-co-sebacate)), PGSA (poly(glycerol-co-sebacate)-acrylate), PLGA (Poly Lactic-co-Glycolic Acid), SEM (scanning electron microscopy), SMG (submandibular gland), SLG (sublingual gland), UV (ultraviolet), VEGF (vascular endothelial growth factor), VEGFR2 (vascular endothelial growth factor receptor 2), XPS (X-ray photoelectron spectroscopy)